DNA structure specificity of Rap endonuclease.

The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.     

The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I

Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence.     

Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA.

In eukaryotic cells, a 5' flap DNA endonuclease activity and a ds DNA 5'-exonuclease activity exist within a single enzyme called FEN-1 [flap endo-nuclease and 5(five)'-exo-nuclease]. This 42 kDa endo-/exonuclease, FEN-1, is highly homologous to human XP-G, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1. These structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap, and its derivative called a pseudo Y-structure. FEN-1 is essential for lagging strand DNA synthesis in Okazaki fragment joining. FEN-1 also appears to be important in mismatch repair. Here we find that human PCNA, the processivity factor for eukaryotic polymerases, physically associates with human FEN-1 and stimulates its endonucleolytic activity at branched DNA structures and its exonucleolytic activity at nick and gap structures. Structural requirements for FEN-1 and PCNA loading provide an interesting picture of this stimulation. PCNA loads on to substrates at double-stranded DNA ends. In contrast, FEN-1 requires a free single-stranded 5' terminus and appears to load by tracking along the single-stranded DNA branch. These physical constraints define the range of DNA replication, recombination and repair processes in which this family of structure-specific nucleases participate. A model explaining the exonucleolytic activity of FEN-1 in terms of its endonucleolytic activity is proposed based on these observations.     

Cloning, expression, and purification of gene 3 endonuclease from bacteriophage T7

The structural gene for the single-stranded endonuclease coded for by gene 3 of bacteriophage T7 has been cloned in pGW7, a derivative of the plasmid pBR322, which contains the lambda PL promoter and the gene for the temperature-sensitive lambda repressor, cI857. The complete gene 3 DNA sequence has been placed downstream of the PL promoter, and the endonuclease is overproduced by temperature induction at mid-log phase of Escherichia coli carrying the recombinant plasmid pTP2. Despite the fact that cell growth rapidly declines due to toxic effects of the excess endonuclease, significant amounts of the enzyme can be isolated in nearly homogeneous form from the induced cells. An assay of nuclease activity has been devised using gel electrophoresis of the product DNA fragments from DNA substrates. These assays show the enzyme to have an absolute requirement for Mg(II) (10 mM), a broad pH optimum near pH 7, but significant activity from pH 3 to pH 9, and a 10-100-fold preference for single-stranded DNA (ssDNA). The enzyme is readily inactivated by ethylenediaminetetraacetic acid or high salt. The differential activity in favor of ssDNA can be exploited to map small single-stranded regions in double-stranded DNAs as shown by cleavage of the melted region of an open complex of T7 RNA polymerase and its promoter.     

A novel structure-specific endonuclease activity associated with polypyrimidine-tract binding (PTB) related protein from rat testis

Nucleases are involved in the processing of various intermediates generated during crucial DNA metabolic processes such as replication, repair, and recombination and also during maturation of RNA precursors. An endonuclease, degrading specifically single-stranded circular DNA, was identified earlier in rat testis nuclear extract while purifying a strand-transfer activity. We are now reporting the purification of this endonuclease, which is a monomeric 42 kDa protein, from rat testis to near-homogeneity. In addition to degrading single-stranded circular DNA, it nicks supercoiled plasmid DNA to generate relaxed DNA and does not act on linear single-stranded or double-stranded DNA. It also makes specific incisions at the single-strand/duplex junction of pseudo-Y, 3'- and 5'-overhangs and 3'- and 5'-flap structures. Other structures such as mismatch, insertion loop, and Holliday junction are not substrates for the testis endonuclease. In contrast to FEN1, the testis endonuclease makes asymmetric incisions on both strands of the branched structures, and free single-stranded ends are not necessary for the structure-specific incisions. Neither 5'-3' nor 3'-5' exonuclease activity is associated with the testis endonuclease. The amino acid sequences of tryptic peptides of the 42 kDa endonuclease show near-identity to polypyrimidine-tract binding protein (PTB) that is involved in the regulation of splicing of eukaryotic mRNA. The significance of the results on the association of structure-specific endonucleae activities with PTB-related protein is discussed.     

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence.

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.     

The specificity of lambda exonuclease. Interactions with single-stranded DNA.

The lambda exonuclease, an enzyme that has been implicated in genetic recombination, rapidly and processively degrades native DNA, starting at the 5' terminus. The enzyme will also degrade the 5'-terminated strand at a single-stranded branch. The experiments reported here reveal various interactions of the enzyme with single-stranded DNA. The rate of digestion is related inversely to the length of single strands. Chains of 100 nucleotides are digested at about 10% the rate of digestion of native DNA. Digestion of the single-stranded ends of lambda DNA does not appear to occur processively. The enzyme binds to circular as well as linear single strands and the affinity for single strands is also related inversely to the chain length. In an equimolar mixture of single- and double-stranded DNA the action of lambda exonuclease on the latteris about half-inhibited. At 20 degrees the initiation of digestion at the 5' terminus of duplex DNA is blocked sterically when such DNA has 3'-terminal single strands that are longer than 100 nucleotides. Information about these properties is important for the practical use of lambda exonuclease as well as for reflections on the role of the enzyme in genetic recombination.     

Interactions of human rad54 protein with branched DNA molecules

The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, approximately 10 +/- 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination.     

Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B, Rad51C, Rad51D and Xrcc2 protein complex

The Rad51B, Rad51C, Rad51D and Xrcc2 proteins are Rad51 paralogs, and form a complex (BCDX2 complex) in mammalian cells. Mutant cells defective in any one of the Rad51-paralog genes exhibit spontaneous genomic instability and extreme sensitivity to DNA-damaging agents, due to inefficient recombinational repair. Therefore, the Rad51 paralogs play important roles in the maintenance of genomic integrity through recombinational repair. In the present study, we examined the DNA-binding preference of the human BCDX2 complex. Competitive DNA-binding assays using seven types of DNA substrates, single-stranded DNA (ssDNA), double-stranded DNA, 5′- and 3′-tailed duplexes, nicked duplex DNA, Y-shaped DNA and a synthetic Holliday junction, revealed that the BCDX2 complex preferentially bound to the two DNA substrates with branched structures (the Y-shaped DNA and the synthetic Holliday junction). Furthermore, the BCDX2 complex catalyzed the strand-annealing reaction between a long linear ssDNA (1.2 kb in length) and its complementary circular ssDNA. These properties of the BCDX2 complex may be important for its roles in the maintenance of chromosomal integrity.